The long-term goal of this research project is to define the specificity, mechanism, and in vivo role of the ubiquitin- dependent protein degradation system which is universally present in eucaryotic cells. This soluble multi-enzyme system has been best characterized from reticulocytes. In that cell it appears to play a role in the degradation of abnormal proteins and in the degradation of intracellular organelles during maturation. A temperature-sensitive mutant of this system is cell cycle arrested, and it therefore must play an important role in differentiation and growth as well. Several significant hemopoetic disease states may be influenced by this system. A number of thallasemias result in synthesis and accumulation of excess globin chains. If this abnormal accumulation of protein could be relived by degradation of the globin, many of the clinical manifestations of the disease might be ameliorated. Secondly, sickle cell anemia can be characterized as a disease which results in the accumulation of abnormal globin chains. The observed in vitro specificity of this system suggests that it may play a role in the degradation of such proteins. Finally, leukemia and other hemopoetic cancers may be thought of as a defect in the programmed maturation and differentiation of progenitor cells. Ubiquitins role in chromatin structure and control of the cell cycle is perhaps involved here too. The in vivo compartmentalization and accessibility of these affected cell types makes these diseases attractive candidates for biochemical intervention. This proposal is designed to provide a basic biochemical description of the specificity and mechanism of this important protein degradation system. Ubiquitin is conjugated to a large number of cellular proteins and it is these carboxy1-terminal conjugates which are thought to be substrates for the proteolytic enzymes of the system. Thus, ubiquitin acts as a signal sequence which is attached of cellular proteins to mark them for degradation. The role of the ubiquitin cofactor will be investigated with natural and chemically modified ubiquitin derivatives. A variety of ubiquitin conjugates will be synthesized and used as substrates in this system. Methods of assay will be developed and the enzymes which metabolize carboxy1-terminal derivatives will be purified and characterized. Finally, inhibitors of ubiquitin activation will be synthesized to probe the role of this system.